Excimer lasers are used for photorefractive surgery and for therapeutic indications.1 The 193 nm ultraviolet light interacts with the tissue through photoablation2, creating a corneal wound. With changes in the beam diameter, beam movement, and the number of exposures, various profiles can be sculptured out of the corneal tissue. This basic principle for the creation of the wound is the same for all excimer lasers and has recently been reviewed in the literature.1 It is, however, the postoperative period and the control of wound healing that determine clinical outcomes, and the limiting factor in predicting these outcomes3 is the lack of detailed understanding of basic events taking place at the surface of the cornea after excimer laser exposure. Predictability of postoperative refractive stability following photorefractive keratectomy (PRK) is still problematic.4,5 Although recent studies suggest that the epithelial cytoarchitecture itself is not altered after PRK using large ablation zone diameters (unpublished data, Hamberg-Nyström, 1996), epithelial hyperplasia is still seen in some patients.6,7 This may, in part, contribute to refractive alterations, especially severe myopic regression. While wound healing events after PRK are the subject of ongoing research, other studies have evaluated the possible effects of laser-associated parameters on wound healing, such as expanding and constricting apertures8 or cellular reactions to excimer laser operations.9"17 Corneal reepithelialization, however, is still the first visible sign of healing and is of significant clinical importance. Corneal erosions created by laser operations increase the risk of corneal infection and are associated with severe pain. Postoperative pain is, in fact, the most immediate and prominent side effect of excimer laser surgery; it can be controlled with topical medications such as diclofenac.18 Additional reports about delayed reepithelialization19 indicate differences in corneal wound healing events between mechanical wounds (such as epithelial scraping) and excimer laser-created wounds. According to our own clinical practice using various excimer lasers, wound healing is not apparently different between lasers. To investigate these empirical findings, rabbits were treated with photorefractive keratectomy employing the two most commonly used excimer lasers: the VISX 20/20 laser (Alcon Inc. Fort Wort, Texas) and the Summit laser (Summit Technology Ine, Waltham, Mass). Epithelial wound healing was monitored and compared.
MATERIALS AND METHODS
For this nonblinded study, 16 albino female New Zealand rabbits, body weight approximately 2.5 kg were used according to the 1987 ARVO Resolution for the use of animals in research. Prior to excimer laser surgery, all animals underwent general anesthesia by intramuscular injection of a ketalar-xylazine mixture.11 Additional topical anesthesia was performed with oxybuprenocain. Surgery was performed on one eye of each rabbit. The surgery was performed with the rabbit in the same position for each procedure. Thorough examination of the external eye was performed before every procedure and no decentration was present.
The epithelium was left in place before surgery. The nictitating membrane was held out of the operation site by the use of forceps and the laser application was in the center of the cornea. Surgery was performed in groups. Group I: Eight rabbits (eight eyes) were treated with Summit Excimed 200 UV LA laser . The wound size was 5 mm in diameter, and all corrections were programmed for -6.00 spherical diopters (D). Group II: Eight rabbits (eight eyes) were treated with VISX 20/20 laser system. The wound size was 5 mm in diameter and all corrections were programmed for -6.00 spherical diopters. All laser treatments were performed without mechanical epithelial abrasion prior to irradiation. No postoperative opthalmic medications were given.
The first measurement of the wound was performed 4 hours after surgery to evaluate possible retraction of the epithelial wound edge. Other parameters of epithelial wound healing were evaluated every 4 hours using photography with fluorescein for staining. Documentation ended after 64 hours when two consecutive examinations demonstrated complete healing with epithelial closure. All photographs were taken with a single lens reflex camera (Nikon, Japan) with Kodak Ektachrome slide film (200 ASA); exposure time 1/60 sec. The wavelength for fluorescein excitation was adjusted by the use of a Wratten gelatin filter no. 47. Every 4 hours, the length of the wound edges were measured with a Run Mate pen (Run Mate, Voubrugge, Holland) on the printed wound edge and the area of the wounds was measured from the projected slides using a standardized magnification system. Additionally, the size and shape of the epithelial wound was monitored at 4, 16, 28, 38, 46, 51, and 54 hours after surgery for each rabbit using a technique known as planometry- a method of calculating the area of a figure (in this case, a wound) with irregular borders, using a specifically designed instrument- a planometer. These calulations were then translated into pictures using a computerized drawing program (Charisma 4.0, Micrografxs, Houston, TX).
Two rabbits, one from each laser group, were excluded from the study due to corneal infection that occurred 8 hours after surgery.
Since the data did not create a normal distribution, a non-parametric test was used to analyze the results. The Mann-Whitney test was applied if there were any differences between groups in wound edge length, wound size, wound closure time, wound edge progress, and wound edge migration velocity.
The early postoperative wound edge length was 18.3 mm for the Summit laser and 16.7 mm for the VISX laser (Table 1). Four hours after surgery, the wounds created by both lasers were slightly but not significantly larger than the theoretical value expected at time 0 for a 5 mm wound. In the final analysis, there was no statistically significant difference in the wound size between the VISX and the Summit groups (Table 1, Fig 1).
The median wound closure time was 53.4 hours for the Summit and 54.0 hours for the VISX laser (Table 1). This difference was not statistically significant.
The pattern of wound edge progression over the wound surface was similar in the two groups (Fig 2), although wound healing progress showed less variance between individual animals within the Summit laser treated group.
Figure 1 : Median epithelial wound are after a 5-mm diameter PRK in rabbits.
Epithelial Wound Closure after a 5-mm Diameter PRK in Rabbits
In a majority of patients, corneal reepithelialization after PRK is achieved after 2 to 3 days.20·21 This is consistent with the wound healing velocity observed after PRK in rabbits.11 The wound healing velocity in our study was similar and did not show any significant laser-dependent difference. The cellular migration velocity increased over time due to the gradual release and accumulation of stimulatory substances from the cells and the wound edge. This can be explained by the fact that cells involved in wound healing, located at the wound edge, are known to promote corneal reepithelialization by the release of various substances, such as growth factors.22,23
In the evaluation of wound healing, the shape of the wound must be considered. The similarity found between treatment groups in the concentric decrease of wound size suggest that the basic mechanism of cellular attachment and interaction during the initial reepithelialization does not seem to be altered either by the use of the excimer laser, or by the specific laser type. All reepithelialization patterns followed the basic pattern of corneal epithelial wound healing described by Buck.24
Although no significant variation existed in the reepithelialization processes between the groups, a slightly larger variation in wound size was noted during epithelial wound healing in the VISX group. A possible explanation may be found in the profile of the wound- especially the shape of the wound edge created by this particular laser. This could be a decisive variable in predicting initial onset and subsequent progress of epithelial regeneration. The shape of the wound edge, in turn, could be influenced by the energy distribution or beam homogeneity in the laser. The difference between the lasers in beam homogeneity in this study, however, did not result in any significant difference in the wound healing events observed.
Future studies need to investigate whether excimer laser surgery influences the basic events within and surrounding cell migration, and therefore influences the integrity of the healed corneal tissue. Better knowledge of these underlying processes may lead to a greater ability to accelerate certain events in the reepithelialization process. Furthermore, it may contribute to a better understanding of other studies investigating the processes which follow primary reepithelialization and lead to multi-layering of the corneal epithelium and the establishment of firm contact to the exposed corneal stroma.25
Figure 2: Drawings of epithelial wound healing patterns after a 5-mm PRK in rabbits.
Migration Velocity of Corneal Epithelium after a 5-mm PRK in Rabbits
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